Broadening The Substrate Scope of Aldolases Through Metagenomic Enzyme Discovery.

Bio-processes based on enzymatic catalysis play a major role in the development of green, sustainable processes, and the discovery of new enzymes is key to this approach. In this work, we analysed ten metagenomes and retrieved 48 genes coding for deoxyribose-5-phosphate aldolases (DERAs, EC 4.1.2.4) using a sequence-based approach. These sequences were recombinantly expressed in Escherichia coli and screened for activity towards a range of aldol additions. Among these, one enzyme, DERA-61, proved to be particularly interesting and catalysed the aldol addition of furfural or benzaldehyde with acetone, butanone and cyclobutanone with unprecedented activity. The product of these reactions, aldols, can find applications as building blocks in the synthesis of biologically active compounds. Screening was carried out to identify optimized reaction conditions targeting temperature, pH, and salt concentrations. Lastly, the kinetics and the stereochemistry of the products were investigated, revealing that DERA-61 and other metagenomic DERAs have superior activity and stereoselectivity when they are provided with non-natural substrates, compared to well-known DERAs.

Realizing the Continuous Chemoenzymatic Synthesis of Anilines Using an Immobilized Nitroreductase.

The use of biocatalysis for classically synthetic transformations has seen an increase in recent years, driven by the sustainability credentials bio-based approaches can offer the chemical industry. Despite this, the biocatalytic reduction of aromatic nitro compounds using nitroreductase biocatalysts has not received significant attention in the context of synthetic chemistry. Herein, a nitroreductase (NR-55) is demonstrated to complete aromatic nitro reduction in a continuous packed-bed reactor for the first time. Immobilization on an amino-functionalized resin with a glucose dehydrogenase (GDH-101) permits extended reuse of the immobilized system, all operating at room temperature and pressure in aqueous buffer. By transferring into flow, a continuous extraction module is incorporated, allowing the reaction and workup to be continuously undertaken in a single operation. This is extended to showcase a closed-loop aqueous phase, permitting reuse of the contained cofactors, with a productivity of >10 gproduct gNR-55-1 and milligram isolated yields >50% for the product anilines. This facile method removes the need for high-pressure hydrogen gas and precious-metal catalysts and proceeds with high chemoselectivity in the presence of hydrogenation-labile halides. Application of this continuous biocatalytic methodology to panels of aryl nitro compounds could offer a sustainable approach to its energy and resource-intensive precious-metal-catalyzed counterpart.

Six-membered cyclic carbonates from trimethylolpropane: Lipase-mediated synthesis in a flow reactor and in silico evaluation of the reaction.

Six-membered cyclic carbonates with hydroxyl and methoxycarbonyloxy functional groups were prepared by transesterification of trimethylolpropane (TMP) with dimethylcarbonate (DMC) by solvent-free lipase-mediated flow reaction followed by thermal cyclization. The flow reaction efficiency was evaluated using different configurations of reactor consisting of packed beds of Novozym®435 (immobilized Candida antarctica lipase B-CalB-a.k.a. N435) and molecular sieves, flowrate, and biocatalyst loads. The mixed column of the biocatalyst and molecular sieves, allowing rapid and efficient removal of the by-product-methanol-was the most efficient setup. Higher conversion (81.6%) in the flow reaction compared to batch process (72%) was obtained using same amount of N435 (20% (w/w) N435:TMP) at 12 h, and the undesirable dimer and oligomer formation were suppressed. Moreover, the product was recovered easily without extra separation steps, and the biocatalyst and the molecular sieves remained intact for subsequent regeneration and recycling. The reaction of CalB with DMC and the primary transesterification product, monocarbonated TMP, respectively, as acyl donors was evaluated by in silico modeling and empirically to determine the role of the enzyme in the formation of cyclic carbonates and other side products. DMC was shown to be the preferred acyl donor, suggesting that TMP and its carbonated derivatives serve only as acyl acceptors in the lipase-catalyzed reaction. Subsequent cyclization to cyclic carbonate is catalyzed at increased temperature and not by the enzyme. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:375-382, 2017.

Multi-steps green process for synthesis of six-membered functional cyclic carbonate from trimethylolpropane by lipase catalyzed methacrylation and carbonation, and thermal cyclization.

A highly functionalized six-membered cyclic carbonate, methacrylated trimethylolpropane (TMP) cyclic carbonate, which can be used as a potential monomer for bisphenol-free polycarbonates and isocyanate-free polyurethanes, was synthesized by two steps transesterifications catalyzed by immobilized Candida antarctica lipase B, Novozym(®) 435 (N435) followed by thermal cyclization. TMP was functionalized as 70 to 80% selectivity of mono-methacrylate with 70% conversion was achieved, and the reaction rate was evaluated using various acyl donors such as methacrylic acid, methacrylate-methyl ester, -ethyl ester, and -vinyl ester. As a new observation, the fastest rate obtained was for the transesterfication reaction using methacrylate methyl ester. Byproducts resulted from leaving groups were adsorbed on the molecular sieves (4Å) to minimize the effect of leaving group on the equilibrium. The difference of reaction rate was explained by molecular dynamic simulations on interactions between carbonyl oxygen and amino acid residues (Thr 40 and Glu 157) in the active site of lipase. Our docking studies revealed that as acyl donor, methyl ester was preferred for the initial conformation of the first tetrahederal intermediate with hydrogen bonding interactions. TMP-monomethacrylate (TMP-mMA) cyclic carbonate was obtained in 63% yield (74.1% calculated in 85% conversion) from the lipase-catalyzed carbonation reaction of TMP-mMA with dimethylcarbonate, and followed by thermal cyclization of the monocarbonate at 90°C. From the multiple reactions demonstrated in gram scale, TMP-mMA cyclic carbonate was obtained as a green process without using chlorinated solvent and reagent.

Kinetic modeling of lipase-catalyzed esterification reaction between oleic acid and trimethylolpropane: a simplified model for multi-substrate multi-product ping-pong mechanisms.

Kinetic models are among the tools that can be used for optimization of biocatalytic reactions as well as for facilitating process design and upscaling in order to improve productivity and economy of these processes. Mechanism pathways for multi-substrate multi-product enzyme-catalyzed reactions can become very complex and lead to kinetic models comprising several tens of terms. Hence the models comprise too many parameters, which are in general highly correlated and their estimations are often prone to huge errors. In this study, Novozym(®) 435 catalyzed esterification reaction between oleic acid (OA) and trimethylolpropane (TMP) with continuous removal of side-product (water) was carried out as an example for reactions that follow multi-substrate multi-product ping-pong mechanisms. A kinetic model was developed based on a simplified ping-pong mechanism proposed for the reaction. The model considered both enzymatic and spontaneous reactions involved and also the effect of product removal during the reaction. The kinetic model parameters were estimated using nonlinear curve fitting through unconstrained optimization methodology and the model was verified by using empirical data from different experiments and showed good predictability of the reaction under different conditions. This approach can be applied to similar biocatalytic processes to facilitate their optimization and design.

Optimization of a two-step process comprising lipase catalysis and thermal cyclization improves the efficiency of synthesis of six-membered cyclic carbonate from trimethylolpropane and dimethylcarbonate.

Six-membered cyclic carbonates are potential monomers for phosgene and/or isocyanate free polycarbonates and polyurethanes via ring-opening polymerization. A two-step process for their synthesis comprising lipase-catalyzed transesterification of a polyol, trimethylolpropane (TMP) with dimethylcarbonate (DMC) in a solvent-free system followed by thermal cyclization was optimized to improve process efficiency and selectivity. Using full factorial designed experiments and partial least squares (PLS) modeling for the reaction catalyzed by Novozym®435 (N435; immobilized Candida antarctica lipase B), the optimum conditions for obtaining either high proportion of monocarbonated TMP and TMP-cyclic-carbonate (3 and 4), or dicarbonated TMP and monocarbonated TMP-cyclic-carbonate (5 and 6) were found. The PLS model predicted that the reactions using 15%-20% (w/w) N435 at DMC:TMP molar ratio of 10-30 can reach about 65% total yield of 3 and 4 within 10 h, and 65%-70% total yield of 5 and 6 within 32-37 h, respectively. High consistency between the predicted results and empirical data was shown with 66.1% yield of 3 and 4 at 7 h and 67.4% yield of 5 and 6 at 35 h, using 18% (w/w) biocatalyst and DMC:TMP molar ratio of 20. Thermal cyclization of the product from 7 h reaction, at 110°C in the presence of acetonitrile increased the overall yield of cyclic carbonate 4 from about 2% to more than 75% within 24 h. N435 was reused for five consecutive batches, 10 h each, to give 3+4 with a yield of about 65% in each run.